Figure 2. Genome integrity of rAAV2/5-CMV-Abca4, -MYO7A, -CEP290, and -EGFP-8.9.

(A) Southern blot analysis of vector DNA isolated directly from rAAV large preparations (2.5 × 10¹⁰ GC/lane) and separated on alkaline agarose gels. Lane 1 contains a marker DNA fragment obtained by restriction digestion from the pAAV2.1-CMV-Abca4 plasmid; lane 2 contains the same DNA fragment as in lane 1 digested with DNaseI, as control of DNaseI activity; lanes 3 and 4: genomes isolated from rAAV2/5-CMV-Abca4; lanes 5 and 6: genomes isolated from rAAV2/5-CMV-MYO7A; lanes 7 and 8: genomes isolated from rAAV2/5-CMV-CEP290; lanes 9 and 10: genomes isolated from rAAV2/5-CMV-EGFP-8.9 (lanes 1–10 belong to the same gel but were noncontiguous); lanes 11 and 12: genomes isolated from rAAV2/5-CMV-EGFP (2.6 kb). Samples in lanes 3, 5, 7, 9, and 11 were treated with DNaseI. (B) Assessment of rAAV2/5-CMV-Abca4 genome length following in vivo delivery. Top panel: Schematic representation of the rAAV2/5-CMV-Abca4 genome with the 2 probes used for the Southern blot analysis. Bottom panel, upper blots: Southern blot analysis of genomic DNA from uninjected muscles (lanes 1 and 3) and an equivalent amount of genomic DNA from murine muscle injected with rAAV2/5-CMV-Abca4 (lanes 2 and 4) digested with NcoI and NotI (lanes 1 and 2) or NcoI alone (lanes 3 and 4). Lanes belong to the same gel but were noncontiguous. The arrows indicate the bands of the expected size. Lower blots: Southern blot analysis with a probe specific for the PDE6B gene used as loading control. Molecular weights are indicated on the left. (C) Western blot analysis with anti-ABCA4 (upper blot) or anti–α-tubulin (lower blot) antibodies of lysates from Cos cells transduced with rAAV2/5. Lane 1: retina from wild-type mouse; lane 2: samples transduced with rAAV2/5-CMV-Abca4; lane 3: samples transduced with rAAV2/5-CMV-EGFP. Anti–α-tubulin was used as loading control. The amounts (μg) of protein loaded are indicated under the respective lanes.